We applied 2D gel electrophoresis and 2D liquid chromatography tandem mass spectrometry (2D LC-MS/MS) to identify proteins that are expressed in
human mesenchymal stem cells (hMSC) undergoing in vitro osteogenic differentiation induced by the osteogenic stimulants (OS) ascorbic acid-2-phosphate,
β-glycerophosphate, and dexamethazone. To assess the effectiveness of OS in inducing osteogenic differentiation, and identify specific changes in protein
expression which accompanied OS treatment, Progenesis workstation software and the Database for Annotation, Visualization and Integrated Discovery
(DAVID) were used classify these proteins and compare them with those found in undifferentiated hMSC and fully differentiated human osteoblasts (hOST).
Of the 534 spots identified in OS-treated hMSC 2D gels, 272 (64%) and 443 (78%) spots matched those found in hMSC and hOST gels, respectively.
Of the 909 different proteins identified by 2D LC-MS/MS in all these cell populations, 144 (16%) were found only in hMSC; 154 (17%) were found only in
OS-treated hMSC; and 121 (13%) were unique to hOST. Differential expression of some of the identified proteins was further confirmed by Western blot
analyses. Significant differences in calcium-mediated signaling molecules and extracellular matrix components were found during osteogenic commitment and
differentiation in OS-treated cells. Gene ontology analysis with DAVID revealed that these functionally related proteins may serve as novel biomarkers for
identifying hMSC commitment along osteogenic fates and demonstrate that protein expression profiling is a valuable tool in elucidating the osteogenic
differentiation of hMSC.